1. Field of the Invention
This invention relates to an expression cassette, a recombinant host cell and a process for producing a target protein, in which a recombinant polynucleotide is constructed to encode a fusion protein comprising: (i) an anchoring protein that comprises a N-terminal amino acid sequence of an ice nucleation protein, so that the fusion protein, once expressed in a host cell, is directed by the anchoring protein to be anchored and exposed on the outer membrane of the host cell; (ii) the target protein; and (iii) a self-splicing protein that comprises a first end fused with the anchoring protein and a second end fused with the target protein, wherein the self-splicing protein comprises a N-terminal or C-terminal amino acid sequence of an intein protein at the second end thereof, such that upon an environmental stimulus, the self-splicing protein exerts a self-cleavage at the second end thereof to release the target protein from the fusion protein. The released fusion protein can then be easily recovered by a simple separating treatment such as centrifugation.
2. Description of the Related Art
With the rapid development of biotechnology, the targets of many researches in the field of life science have changed from genes to proteins, and the techniques for protein isolation and purification have attracted great attention from investigators worldwide. Recently, many techniques have been developed to isolate and purify proteins. However, not a few factors, for example the diversity of protein sources (e.g., microbial fermented broths, plant cells, animal cells, urines and bloods), the low concentrations of proteins, and the impurities contained in protein samples, increase the difficulty and complexity of protein isolation and purification. In particular, production of protein in large quantities is usually expensive due to the elaborate procedures and the specialized equipment required for cell disruption, repetitive solid-liquid phase separation, possible column purification involvement, and concentration.
In current protein engineering methods, an effective way to simplify the purification procedure is to produce a fusion protein in which an affinity tag is added to the N- or C-terminus of a target protein of interest (M. T. Hearn and D. Acosta (2001), J. Mol. Recognit., 14:323-369). These kinds of tags can be glutathione S-transferase (GST), maltose-binding protein (MBP), polyhistidine, streptavidin, chitin-binding domain, or a combination thereof. Such tagged-fusion proteins can be recovered by affinity chromatography. The affinity purification procedures specifically designed for the fusion protein tags have been well established in literature (C. Mateo et al. (2001), J. Chromatogr. A, 915:97-106; K. Sakhamuru et al. (2000), Biotechnol. Prog., 16:296-298). However, affinity columns used in affinity purification procedures of proteins are comparatively expensive and only available for small scale production. In addition, in cases of therapeutic protein, tags need to be removed from the fusion proteins. To achieve this, a protease cleavage site such as a factor Xa cleavage site, a tobacco etch virus (TEV) site, etc., was built in the fusion between the tag and the target protein (V. Schauer-Vukasinovic et al. (2002), Anal. Bioanal. Chem., 373:501-507), and a site-specific protease such as the factor Xa protease or the TEV protease, etc., was required to effect a splicing at the protease cleavage site so as to release the target protein (T. D. Parks et al. (1994), Anal. Biochem., 216:413-417). These site-specific proteases, however, are costly and removal of the same involves complicated procedures. Accordingly, lowering the production cost, simplifying the manufacturing procedures while increasing the yield of recombinant proteins, etc., have become the main goals of researchers in the biotech industry.
In the last decade, several self-cleaving protein modules have been developed and combined with conventional affinity tags to create new and simple affinity purification methods. In particular, a number of engineered self-cleaving inteins have been successfully used in bioseparation processes. In practice, the self-cleaving reaction can be induced at the intein's N-terminus by thiol addition or its C-terminus by a mild pH shift. The pTWIN vectors and the IMPACT™ system from New England Biolabs are the most published commercial intein systems to date, and are often paired with a chitin-binding domain as the affinity tag. A majority of the NEB systems are based on thiol-induced inteins, which can be induced by compounds including 2-mercaptoethane sulfonic acid (MESNA), hydroxylamine, thiophenol, β-mercaptoethanol, 1,4-dithiothreitol (DTT) or free cysteine. Typically, 15-30 mM DTT addition is used to trigger the cleaving reaction for N-terminally cleaving inteins. This concentration of DTT will generally reduce disulfide bonds in proteins containing them, effectively inactivating those targets. Other compounds such as MESNA, hydroxylamine or free cysteine can also be used as cleaving triggers, but they tend to leave modifications at the C-terminus of the target protein, which could affect product activity in some cases. Therefore, for disulfide-bond-containing protein targets, the thiol-induced inteins are not ideal unless the target protein can maintain activity after thiol treatment and extra modifications at the C-terminus can be tolerated (Wan-Yi Wu et al. (2011), Protein Expression and Purification, 76:221-228).
An efficient C-terminal cleaving intein is the DI-CM intein derived from the Mycobacterium tuberculosis recA intein. It is 18 kDa in size, and has been paired with conventional affinity tags as well as non-chromatographic purification tags. Compared to the DTT-induced inteins, the cleaving activity of the ΔI-CM intein is induced by a mild pH change from pH 8.5 to pH 6.0-6.5, suggesting its compatibility with disulfide-bonded targets. The ΔI-CM intein is also temperature dependent, allowing the purification conditions to be adjusted according to the needs of each specific target (Wan-Yi Wu et al. (2011), supra).
The IMPACT™ system from New England Biolabs is not ideal for industrial use since it requires the use of a costly chitin column, which is unsuitable for the large scale production of target proteins.
Amongst various techniques for protein isolation and purification known in the art, microbial surface display systems, which could lower production cost and simplify manufacturing procedure, have been considered to have potential for use in protein engineering. Generally, the microbial surface display system is composed of a carrier protein (also called anchoring motif), a passenger protein (i.e., the target protein), and the host cell. The carrier proteins normally are cell surface proteins associated with signal transduction, surface adherence, cell-cell recognition and immunoreaction, and those for ion channels for molecule transport. Commonly used carrier proteins include bacterial fimbriae, S-layer proteins, ice nucleation protein (INP), and outer membrane proteins (Sang Yup Lee et al. (2003), TRENDS in Biotechnology, 21:45-52; Po-Hung Wu et al. (2006), Biotechnology and Bioengineering, 95:1138-1147). Target protein can be fused with the carrier protein via N-terminal fusion, C-terminal fusion or sandwich fusion to the form a fusion protein, which, once expressed, can be displayed on the host cells' surface.
For example, US 2005/0015830 A1 discloses a process of producing a protein or polypeptide of interest in a plant or in plant cells, comprising: (i) transforming or transfecting a plant of plant cells with a nucleotide sequence having a coding region encoding a fusion protein comprising the protein or polypeptide of interest, a signal peptide functional for targeting said fusion protein to the apoplast, and a polypeptide capable of binding the fusion protein to a cell wall component, (ii) enriching cell wall components having expressed and bound fusion protein, and (iii) separating the protein or polypeptide of interest or a protein comprising the protein or polypeptide interest. Particularly, the protein or polypeptide of interest may contain one or more affinity peptide tags, such as an intein or part thereof. According to US 2005/0015830 A1, step (iii) involves cleavage of at least one peptide bond. Therefore, said fusion protein may further comprise a cleavage sequence allowing cleavage of the fusion protein, wherein the cleavage of the fusion protein may be achieved by intein-mediated cleavage. However, it is somewhat difficult to culture plant cells as compared to microbial cells. In addition, an enriching step is required so as to increase the concentration of cell wall components having expressed and bound fusion protein. Besides, the product obtained from the separating step contains not only the purified protein or polypeptide of interest but also other protein components such as signal peptide. Therefore, US 2005/0015830 A1 fails to provide a rapid and cost-efficient technique for the production of target proteins.
TW I304810 discloses an oil body-based purification method for proteins, comprising the steps of: (a) preparing a polypeptide comprising oil body-binding oleosin, an intein connected to the oleosin and a target protein connected to the intein, wherein the intein is Mxe GyrA or Ssp DnaB; (b) mixing the polypeptide with oil body so as to form an oil body mixture; (c) separating the oil body mixture from the extracted cell debris; (d) cleaving the polypeptide from the oil body mixture; and (e) separating the polypeptide from the rest of the oil body mixture. According to TW I304810, the oleosin acts as a carrier protein and the intein is Mxe GyrA or Ssp DnaB. However, liquid oil must be used in the mixing and separating steps, which inevitably increases process complexity and manufacturing cost.
Ice nucleation protein (INP) is an outer membrane protein (OMP) found in several plant pathogenic bacteria, namely Pseudomonas (e.g., Pseudomonas syringae, Pseudomonas borealis, Pseudomonas putida), Xanthomonas (e.g., Xanthomonas campestris) and Erwinia (e.g., Erwinia herbicola). INP enables the bacteria to survive freezing through formation of ice on the surface of the bacteria (L. Li et al. (2004), Biotechnol. Bioeng., 85:214-221). INP has several unique structural and functional features that make it highly suitable for use in a bacterial surface display system. The specific amino acids of the N-terminal domain are relatively hydrophobic and link the protein to the outer membrane via a glycosylphosphatidyl inositol anchor. The C-terminal domain of the protein is highly hydrophilic and exposed to the medium. The central part of INP comprises a series of repeating domains that act as templates for ice crystal formation. It has been shown that full-length INP and various truncates that lack the central repeating domain yield stable surface display. This indicates that the central repeating domains are not required for export to the cell surface, and are therefore, ideal spacer units to control the distance between the passenger protein and the cell surface. A derivative that comprises the N- and C-terminal domains of INP is commonly used for surface display. However, the N-terminal domain appears to be the only prerequisite for successful targeting and surface-anchoring. Importantly, INP can be expressed at the cell surface of E. coli at a very high level, without affecting cell viability (Edwin van Bloois et al., Trends in Biotechnology, February 2011, Vol. 29, No. 2, pp. 79-86). By fusing various target proteins to the C-terminus of INP, it was found that the engineered host cell had the surface-localized activities of the target proteins (R. Freudl et al. (1986), J. Mol. Biol., 188:491-494; A. Charbit et al. (1986), EMBO J., 5:3029-3037; H. C. Jung et al. (1998), Enzyme Microb. Technol., 22:348-354; E. J. Kim et al. (1999), Lett. Appl. Microbiol., 29:292-297; W. Bae et al. (2002), J. Inorg. Biochem., 88:223-227; P. H. Wu et al. (2006), Biotechnol. Bioeng., 95:1138-1147). INP has also been used in the microbial cell surface display of levansucrase, carboxymethylcellulase (CMCase), salmobin and organophosphorus hydrolase (OPH) (Sang Yup Lee et al. (2003), TRENDS in Biotechnology, 21:45-52; Po-Hung Wu et al. (2006), Biotechnology and Bioengineering, 95:1138-1147).
In order to develop a new rapid and cost-efficient technique for the massive production of target proteins by recombinant technology, the applicants attempted to create an expression system for target protein in fusion protein form, in which INP is used as a carrier protein for anchoring on the host cell's surface, and intein is used as an intramolecular cleavage site for releasing the target protein, such that the target protein can be easily recovered by a simple separating treatment such as centrifugation.